Development Of Dwarf Apple Plants Via RNAi Suppression Of GA20-ox Gene Expression

Apple （Malns×domestica Borkh.） is one of the most widely cultivated fruit crops world-wide, can become very large which impart the benefits of decreased time to flowering from planting, increased flower number and reduced vegetative vigor. Furthermore, the dwarfing trees make mechanical harvesting more easily that becomes a trend of changing fruit cultivation. Trees can become smaller if grown as scions grafted on to a dwarfing rootstock or interstock. Certain rootstocks impart the benefits of decreased time to flowering from planting, increased flower number and reduced vegetative vigour. For many crops, chemical growth retardants are used as a supplementary treatment to control height of trees. Most growth retardants act by inhibiting the biosynthesis of the gibberellin （GA）-a plant hormones. The GAs biosynthetic pathway has been well characterized and can be divided into three major stages according to the localization and the enzymes involved. GA20-ox, which catalyst the penultimate step in the formation of bioactive GAs, has been shown to be an important regulator in GA biosynthesis pathway. Systemic silencing can spread from rootstocks to scion and vice versa. For verify the hypothetical, we modified plant height via RNAi suppression of MdGA20ox gene expression in Pingyitiancha and Hanfu （a type of apple with higher transformation rate）. The aim of the work was to reveal the impact of the GA20-ox genes on plant growth and height. The main results are as follows:1. In the subculture stage, suitable medium for the proliferation of Pingyitiancha was MS+1 mg.L-’6-BA+0.1 mg·L_-1NAA+0.2 mg.L_-1 GA, the multiplication coefficient was 3.37. Suitable medium for the rooting was 1/2MS+0.6 mg.L_-1 IBA+0.5g.L_-1 AC, and kept in dark for 5 days, rooting rate was 69.67%. On the MS+IAA 0.3 mg.L_-1+BA 1.0 mg.L_-1, the survival rate of Hanfu/Pingyitiancha was 80% with 2 leave on the scions.2. High adventitious shoot regeneration system from leaves in vitro of Pingyitiancha apple was obtained,the best medium suiting leaves regeneration of the apple in vitro was MS+1 mg.L_-16-BA+0.1 mg.L_-1NAA+0.2 mg.L_-1GA. Other factors of regeneration system are: regeneration rate of paraxial cotyledons are higher than abaxial cotyledons; the explants were kept in darkness about 3 weeks which are the middle parts of the top leaves from the plant cultured about 21 d; leave of Pingyitiancha were sensitive to Kan. All stems turn white in 30 d when the concentration of Kan was 25 mg.L_-1, then died 10 days later; GA can promote plants’heigh, and the suitable concentration is 0.5 mg.L_-1.3. A fragment of 466 bp for GA20-ox gene obtained through PCR amplification both in Hanfu and Pingyitiancha that showed 100% homologies in nucleotide sequence when comparing the two fragments with published sequence of apple that used to make an hpRNA construct, and named MdGA20ox. The conserved region of 466 bp fragment and the binary vector pKANNBAL and pART27 were used to make an hpRNA construct to silence GA20-ox in apple. To create an inverted repeat construct RNAi, the EcoR I restriction site at the 5-and Xho I restriction site at the 3-end of the fragment were incorporated through PCR using the forward primer pF2 and reverse primer pR2 for cloning the fragment in sense orientation between the 35 S promoter and intron. While the Hind III restriction site at the 5’-end and the Xba I restriction site at the 3’-end of the fragment were incorporated through PCR using the forward primer pF3 and the reverse primer pR3 to clone the fragment in anti-sense orientation, named pRNAi-GA20ox.4. Suppression of GA20-ox resulted in shorter stems, a decreased length of inter-nodes. All the transgenic lines are lower than the non-transgenic control, for example, in T-HF2, the lowest transgenic line, height is only 57% of the wild. The inter-nodes number of the transgenic line is 1.3-fold than that of the control （4.3/3.4） on average, with the average inter-nodes length of 0.44 cm. Surprisingly, multiplication rates of transgenic lines are increased compared with the non-transgenic lines at the meantime. These results demonstrate that the GA20-ox plays an important role in control of vegetative growth. Transgenic root stocks （T-HF2 and T-PT3） can also affect the non-transgenic scion （GL） in the micro grafting. Inter-nodes length and the inter-nodes number of GL/T-HF2 （4.53,0.63 cm） and GL/T-PT3 （4.83,0.61 cm） were higher than control （3.00,1.00 cm）...